Calculation of reference values for and application of color deconvolution. (A) Histogram of log2 4', 6-diamidino-2-phenylindole (DAPI)/propidium iodide (PI) signal ratios from 897 points (single pixels) automatically identified from 3 fields of view of procyclic Trypanosoma brucei stained with DAPI and PI. Each point samples a single kinetoplast or nucleus except in rare cases (<5%) where multiple points were identified per organelle. Points were automatically assigned to either a high log2 ratio (536 points) or a low log2 ratio (361 points) group. (B) An example region of one of the source DAPI fluorescence micrographs used to generate the data in (A). Each point analyzed is marked with a ring color coded to indicate whether it fell into the high (magenta) or low (green) log2 ratio group. Scale bar represents 10 μm. (C) DAPI and PI values of the 897 example points. Points were identified as nuclei or kinetoplasts as described in (A) and (B). The average DAPI and PI signal for kinetoplasts and nuclei, which are the reference values for color deconvolution, are indicated. (D) The kinetoplast and nuclear signal values of the same 897 points following color deconvolution. Color deconvolution acts, using the reference values, to maximize the separation of signal from kinetoplasts and nuclei.
Wheeler et al. BMC Biology 2012 10:1 doi:10.1186/1741-7007-10-1